RiboTag: A novel technique to profile cell type specific gene expression and inv

RiboTag：一种分析细胞类型特异性基因表达和反转录的新技术

• 批准号: 7935264
• 负责人: PAUL S AMIEUX
• 金额: $ 38.61万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935264
• 关键词: Address; Aging; Antibodies; Biochemical; Biochemical Genetics; Biological Assay; Brain; Cell Nucleus; DNA Sequence; DNA Sequence Analysis; Disease; Environment; Epitopes; Event; Gene Expression; Gene Proteins; Genes; Genetic Techniques; Genetic Translation; Goals; Hormones; Immunologic Techniques; Immunoprecipitation; In Situ Hybridization; Individual; Lasers; Learning; Measures; Memory; Messenger RNA; Methods; Microarray Analysis; Molecular Profiling; Mouse Strains; Mus; Nervous system structure; Neuraxis; Neuromodulator; Neurons; Neurosciences; Neurotransmitters; Occupations; Organism; Pharmaceutical Preparations; Physiological; Polyribosomes; Population; Protein Isoforms; Proteins; RNA; Regulation; Reporter; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Proteins; Ribosomes; Specificity; Speed; Stress; Substance abuse problem; Synapses; Synaptic plasticity; Techniques; Technology; Testing; Time; Transgenic Organisms; Translating; Translational Regulation; addiction; cell type; drug of abuse; improved; in vivo; nervous system disorder; new technology; next generation; novel; protein expression; public health relevance; recombinase; response; tool

DESCRIPTION (provided by applicant): The brain is composed of hundreds of different neuronal subtypes that each have unique jobs to do. One of the most significant long-term goals of neuroscience is to understand how these neuronal subtypes do their job by producing specific proteins, contacting other neurons, and changing in response to physiological and pathological contexts. Unfortunately, techniques to capture the total translated mRNA from a defined subtype of neurons as the organism responds to the environment, hormones, drugs, and other regulators are lacking. This proposal addresses that need with a novel biochemical and genetic technique to express epitope-tagged ribosomal proteins in response to a cell type specific Cre recombinase in mouse brain. The polyribosomes containing the transcribed and translatable mRNAs are isolated by immunological techniques and then the RNA is analyzed by PCR, microarray, and next-generation DNA sequencing. Our specific aims are to: (1) Develop techniques to optimize polyribosome immunoprecipitation from brain using the RiboTag mouse that has already been created with an HA-tagged Rpl22 ribosomal protein under Cre recombinase regulation. (2) Create a new RiboTag mouse line with a Flag-tagged Rpl23a that will respond to Cre recombinase activation. (3) Activate Rpl22-HA and Rpl23a- Flag with specific Cre recombinase transgenics to test the ability of the RiboTag isolation technique to detect neuron specific mRNAs and the changes that occur under in vivo physiological regulation. (4) Examine whether the RiboTag techniques differentiate between translated mRNAs and those that are translationally repressed. These novel mouse strains and biochemical methods should allow neuroscientists to measure changes in gene expression and translational regulation with greater resolution and higher throughput than previously available and speed our understanding of the underlying changes in RNA and protein that determine synaptic plasticity. In order to understand how the brain adapts to a changing environment under physiological circumstances or how specific neuronal populations degenerate or malfunction in disease, we must have robust and widely available techniques to measure gene and protein expression in specific neuronal subtypes. The ribosome tagging (RiboTag) technology we are developing will provide such a tool to address questions about gene expression and mRNA translation during adaptive changes in the brain-changes that occur during memory and learning, substance abuse and addiction, aging, and in response to neurological disorders. PUBLIC HEALTH RELEVANCE: In order to understand how the brain adapts to a changing environment, we must understand how genes are regulated in real time in specific neuronal circuits and nuclei in the brain. If successful, the ribosome tagging technology will provide a new technology to address questions about how genes are regulated during adaptive events in the brain-events such as memory consolidation, adaptation to drugs of abuse, and adaptive events related to post traumatic stress.

描述（由申请人提供）：大脑由数百种不同的神经元亚型组成，每个人都有独特的工作要做。神经科学的最重要的长期目标之一是了解这些神经元亚型如何通过产生特定的蛋白质，接触其他神经元并响应生理和病理环境而改变。不幸的是，由于缺乏有机体对环境，激素，药物和其他调节剂的反应，从定义的神经元亚型中捕获总翻译的mRNA的技术。该提案解决了需要一种新型的生化和遗传技术，以对小鼠大脑中细胞类型特异性CRE重组酶的反应表达表位标记的核糖体蛋白。包含转录和可翻译的mRNA的多核糖体通过免疫学技术分离，然后通过PCR，微阵列和下一代DNA测序分析RNA。我们的具体目的是：（1）使用已经在CRE重组酶调节下使用HA标记的RPL22核糖体蛋白创建的Ribotag小鼠开发技术来优化大脑的多氧化肌体免疫沉淀。 （2）使用标记为标记的RPL23A创建新的Ribotag鼠标线，该系列将响应CRE重组酶激活。 （3）用特定的CRE重组酶转基因激活RPL22-HA和RPL23A-FLAG，以测试Ribotag隔离技术检测神经元特异性mRNA的能力以及体内生理调节下发生的变化。 （4）检查Ribotag技术是否会区分翻译的mRNA和被翻译抑制的mRNA。这些新型的小鼠菌株和生化方法应使神经科学家能够以比以前可获得的更大的分辨率和更高的吞吐量测量基因表达和翻译调节的变化，并加快我们对确定突触可塑性的RNA和蛋白质的潜在变化的理解。为了了解大脑在生理环境下如何适应不断变化的环境，或者特定的神经元种群如何在疾病中退化或故障，我们必须具有强大且可广泛的技术来测量特定神经元亚型中的基因和蛋白质表达。我们正在开发的核糖体标记（Ribotag）技术将提供这样的工具，以解决有关在记忆和学习，滥用药物滥用和成瘾，衰老以及对神经系统疾病的响应期间发生的大脑变化过程中基因表达和mRNA翻译问题的问题。公共卫生相关性：为了了解大脑如何适应不断变化的环境，我们必须了解如何在特定的神经元电路和大脑中的特定神经元电路和核中实时调节基因。如果成功，核糖体标记技术将提供一项新技术，以解决有关在脑事件中如何调节基因的问题，例如记忆巩固，适应滥用药物以及与后创伤后压力有关的自适应事件。